L-amino acid producing microorganism having attenuated carbamyl phosphate synthase activity and method for producing L-amino acid using same

Abstract

The present application relates to an L-amino acid-producing microorganism having attenuated carbamoyl phosphate synthase activity, and a method for producing an L-amino acid using the same.

Description

Technical Field

[0001] The present disclosure relates to an L-amino acid-producing microorganism in which the activity of carbamoyl phosphate synthetase is attenuated, and a method for producing L-amino acids using the microorganism. Background Art

[0002] In the process of producing L-amino acids using microorganisms, ATP is an important substance, which is used as a cofactor for biosynthetic enzymes or in various metabolic processes for cell growth and activity maintenance.

[0003] Carbamoyl phosphate is an intermediate metabolite used in the pyrimidine biosynthetic pathway and the arginine biosynthetic pathway. Carbamoyl phosphate synthetase is an enzyme that converts bicarbonate into carbamoyl phosphate, using two molecules of ATP and one molecule of glutamine as nitrogen donors in the production of carbamoyl phosphate. Glutamine is produced by glutamine synthetase using glutamate as a substrate, consuming one molecule of ATP in the process.

[0004] In this regard, it has been reported that when a variant carbamoyl phosphate synthetase in which feedback inhibition is desensitized and high activity is maintained is introduced into Escherichia coli (E. coli), the production of L-arginine, citrulline, and pyrimidine derivatives increases (Patent Document 1).

[0005] However, it has been reported that excessive formation of intracellular carbamoyl phosphate leads to cytotoxicity (Non-Patent Document 1). Carbamoyl phosphate is a very unstable substance, and cyanate naturally converted from carbamoyl phosphate may cause cytotoxicity.

[0006] Therefore, if the activity of carbamoyl phosphate synthetase in microorganisms is controlled to maintain an appropriate intracellular level of carbamoyl phosphate, prevent unnecessary waste of ATP, and control energy balance, it is possible to increase the production of L-amino acids.

[0007] [Prior art literature]

[0008] [Patent Literature]

[0009] (Patent Document 1) JP 2003-093083 A

[0010]

Non-patent documents

[0011] (Non-patent document 1) Charlier et al., Amino Acids. 2018; 50(12): 1647–1661. Summary of the invention

[0012]

Technical issues

[0013] The present inventors have developed a microorganism of the genus Corynebacterium having an L-amino acid-producing ability, in which the activity of carbamoyl-phosphate synthetase is attenuated, a method for producing L-amino acids using the microorganism, a composition for producing L-arginine, the composition comprising the microorganism, and use of the microorganism in producing L-arginine, thereby completing the present disclosure.

[0014]

Technical solution

[0015] An object of the present disclosure is to provide a Corynebacterium microorganism having L-amino acid-producing ability, in which the activity of carbamoyl-phosphate synthetase is attenuated.

[0016] Another object of the present disclosure is to provide a method for producing L-amino acids, the method comprising the step of culturing a microorganism of the genus Corynebacterium having an L-amino acid-producing ability in a culture medium, in which the activity of carbamoyl-phosphate synthetase is attenuated.

[0017] Another object of the present disclosure is to provide a composition for producing L-amino acids, the composition comprising the Corynebacterium microorganism of the present disclosure; a culture medium for culturing the microorganism; or a combination thereof.

[0018] Another object of the present disclosure is to provide a method for preparing a Corynebacterium microorganism having an L-amino acid-producing ability, the method comprising the step of attenuating carbamoyl-phosphate synthetase.

[0019] Another object of the present disclosure is to provide use of a microorganism of the genus Corynebacterium in which the activity of carbamoyl-phosphate synthetase is attenuated in producing L-amino acids.

[0020]

Beneficial Effects

[0021] Compared with existing unmodified microorganisms, the microorganism of the present disclosure can enhance L-amino acid production capacity by attenuating the activity of carbamoyl phosphate synthetase. DETAILED DESCRIPTION

[0022] The present disclosure will be described in detail below. At the same time, each description and embodiment disclosed in the present disclosure can also be applied to other descriptions and embodiments. That is to say, all combinations of the various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific description described below. In addition, many papers and patent documents are referenced and cited throughout the specification. The disclosures of the cited papers and patent documents are incorporated herein by reference as a whole to further illustrate the level and scope of the subject matter to which the present disclosure belongs.

[0023] One aspect of the present disclosure provides a Corynebacterium microorganism having an L-amino acid-producing ability, wherein the activity of carbamoyl-phosphate synthetase is attenuated.

[0024] As used herein, the term "carbamoyl-phosphate synthetase" refers to an enzyme that converts bicarbonate into carbamoyl-phosphate.

[0025] Carbamoyl phosphate synthetase may be composed of a carbamoyl phosphate synthetase small subunit (CarA) and a carbamoyl phosphate synthetase large subunit (CarB), and the amino acid sequence of CarA or CarB may be obtained from a known database such as Genbank of NCBI.

[0026] For example, CarA or CarB of the present disclosure can be derived from a microorganism. The microorganism can be specifically derived from a microorganism of the genus Corynebacterium, more specifically, from Corynebacterium glutamicum, Corynebacterium deserti, Corynebacterium crudilactis, Corynebacterium efficiens, Corynebacterium callunae, etc., but is not limited thereto.

[0027] For another example, the amino acid sequence of CarA disclosed herein may be ANU33813.1 derived from Corynebacterium glutamicum ATCC 13869, and the amino acid sequence of CarB may be ANU33812.1 derived from Corynebacterium glutamicum ATCC 13869, but it is obvious that proteins having CarA or CarB activity derived from various sources may be included.

[0028] In the present disclosure, CarA may have, include, or consist of the amino acid sequence shown in SEQ ID NO: 1, or consist essentially of the amino acid sequence; CarB may have, include, or consist of the amino acid sequence shown in SEQ ID NO: 2, or consist essentially of the amino acid sequence.

[0029] In the present disclosure, the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 2 may include an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity with the amino acid sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2. It is also obvious that any protein having an amino acid sequence with deletion, modification, substitution, conservative substitution or addition in part of the sequence may also fall within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the protein comprising the amino acid sequence SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0030] Examples thereof include sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within the amino acid sequence that do not alter the function of the protein of the present disclosure.

[0031] "Conservative substitutions" refer to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions generally occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0032] As used herein, the term "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or nucleotide sequences, and can be expressed as a percentage. The terms "homology" and "identity" are often used interchangeably.

[0033] The sequence homology or identity of conservative polynucleotides or polypeptides is determined by standard comparison algorithms, and the default gap penalty set up by the used program can be used together. Basically, homologous or identical sequences are usually able to hybridize with all or part of the sequence under medium or high stringency conditions. Obviously, hybridization also includes hybridization with polynucleotides containing universal codons or codons that take into account codon degeneracy.

[0034] Known computer algorithms (such as the "FASTA" program) can be used to determine whether any two polynucleotide or polypeptide sequences have homology, similarity or identity, for example using the default parameters in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) can be used, which is implemented as follows: the Needleman program (version 5.0.0 or later) in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (including the GCG program package (Devereux, J. et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [et al., J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ET AL., 1996)). Homology, similarity or identity can be determined using BLAST from the National Center for Biotechnology Information, or ClustalW.

[0035] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, the GAP computer program, e.g., Needleman et al. (1970), J Mol Biol. 48:443, as described in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as the value obtained by dividing the number of similar aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program may include (1) a binary comparison matrix (including a value of 1 for identity and a value of 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Acids Res. 14:6745 (or the EDNAFULL (EMBOSS version of NCBINUC 4.4) substitution matrix), as disclosed in Schwartz and Dayhoff, ed., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0036] CarA and CarB of the present disclosure may be encoded by carAB operon genes.

[0037] For example, the carA gene may be a polynucleotide encoding ANU33813.1 derived from Corynebacterium glutamicum ATCC 13869, and the carB gene may be a polynucleotide encoding ANU33812.1 derived from Corynebacterium glutamicum ATCC 13869, but is not limited thereto. Obviously, the present disclosure includes carA and / or carB genes derived from various sources, which encode proteins having CarA and / or CarB activity.

[0038] As used herein, the term "polynucleotide" is a polymer of nucleotides in which nucleotide monomers are linked by covalent bonds into a long chain, and refers to a DNA or RNA chain of a predetermined or longer length, and more specifically, refers to a polynucleotide fragment encoding a protein.

[0039] The polynucleotide encoding the carbamoyl phosphate synthetase of the present disclosure may include a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2. In one embodiment of the present disclosure, the polynucleotide of the present disclosure may have or include the sequence of SEQ ID NO: 3. In addition, the polynucleotide of the present disclosure may consist of the sequence of SEQ ID NO: 3 or consist essentially of the sequence of SEQ ID NO: 3. Specifically, the carbamoyl phosphate synthetase may be encoded by the polynucleotide shown in the nucleotide sequence of SEQ ID NO: 3.

[0040] In the polynucleotides of the present disclosure, various modifications may be made in the coding region, taking into account the degeneracy of codons or preferred codons in an organism intended to express the carbamoyl phosphate synthetase of the present disclosure, as long as the amino acid sequence of the carbamoyl phosphate synthetase is not changed. Specifically, the polynucleotides of the present disclosure may have or include a nucleotide sequence having 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more homology or identity with the sequence of SEQ ID NO: 3, or may consist of or be substantially composed of a nucleotide sequence having 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more homology or identity with the sequence of SEQ ID NO: 3, but are not limited thereto.

[0041] In addition, the polynucleotides of the present disclosure may include probes prepared from known gene sequences, for example, any sequence, as long as it is a sequence that can hybridize with all or part of the complementary sequence of the polynucleotide sequence of the present disclosure under stringent conditions, is not limited. "Stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. These conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). Examples include conditions in which polynucleotides with higher homology or identity, i.e., polynucleotides with homology or identity of 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, whereas polynucleotides with lower homology or identity do not hybridize with each other, or conditions in which washing is performed once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS, which are general washing conditions for Southern hybridization.

[0042] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are allowed depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present disclosure may also include substantially similar nucleic acid sequences and isolated nucleic acid fragments that are complementary to the entire sequence.

[0043] Specifically, the hybridization conditions including the hybridization step with a Tm value of 55° C. and the above conditions can be used to detect polynucleotides having homology or identity with the polynucleotides disclosed herein. In addition, the Tm value can be 60° C., 63° C. or 65° C., but is not limited thereto, and those skilled in the art can make appropriate adjustments according to the purpose.

[0044] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, and these variables are well known in the art (eg, J. Sambrook et al., supra).

[0045] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and it can be a microorganism in which a specific mechanism is weakened or enhanced due to the insertion of exogenous genes or the enhancement or inactivation of the activity of endogenous genes, and it can be a microorganism comprising a genetic modification for producing a target polypeptide, protein or product.

[0046] In one embodiment of the present disclosure, the microorganism of the present disclosure may be a microorganism in which the activity of carbamoyl phosphate synthetase is weakened. The microorganism may be a microorganism in which the activity of any one or more selected from the group consisting of CarA and CarB is weakened. For example, compared with the endogenous activity, the microorganism of the present disclosure may be a microorganism in which CarA and CarB or the carAB operon genes encoding them are weakened, but is not limited thereto.

[0047] As used herein, the term "reduction" of polypeptide (e.g., protein specified by the name of each enzyme) activity has all concepts covering reduced activity or loss of activity compared to endogenous activity. Reduction can be used interchangeably with terms such as inactivation, lack, downregulation, reduction, decrease, weakening, etc.

[0048] Attenuation can also include: the situation where the activity of the polypeptide itself is reduced or disappears compared with the original polypeptide activity of the microorganism due to the variation of the polynucleotide encoding the polypeptide; the situation where the overall polypeptide activity level and / or concentration (expression level) in the cell is lower than that of the natural strain due to the inhibition of gene expression of the polynucleotide encoding the polypeptide or the inhibition of translation into polypeptide; the situation where the polynucleotide is not expressed at all; and / or the situation where there is no polypeptide activity even when the polynucleotide is expressed. "Endogenous activity" refers to the activity of a specific polypeptide originally present in the parent strain or wild-type or unmodified microorganism before the trait is changed when the trait is changed due to genetic variation caused by natural or artificial factors. This can be used interchangeably with "activity before modification". "Attenuation, inactivation, lack, reduction, downregulation, reduction or reduction" of the polypeptide activity compared with its endogenous activity means that the activity of the polypeptide is reduced compared with the activity of the specific polypeptide originally present in the parent strain or unmodified microorganism before the trait is changed.

[0049] For the purpose of the present invention, the microorganism disclosed in the present invention is a microorganism having an improved L-amino acid production ability by having an attenuated carbamoyl phosphate synthetase activity, and the unmodified microorganism having an unattenuated carbamoyl phosphate synthetase, which is a target strain for comparing whether the L-amino acid production ability is enhanced, may be a wild-type Corynebacterium glutamicum ATCC 13869 strain, a Corynebacterium glutamicum ATCC 13869 strain with attenuated ArgF protein activity known as an L-ornithine-producing strain, or a Corynebacterium glutamicum ATCC 13869 strain with attenuated ArgR protein activity, but is not limited thereto.

[0050] The reduction of the activity of such a polypeptide can be carried out by any method known in the art, but is not limited thereto, and can be achieved by applying various methods well known in the art (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014; 15(2): 2773-2793, Sambrook et al., Molecular Cloning 2012, etc.).

[0051] Specifically, attenuating the activity of the polypeptide of the present disclosure can be achieved by:

[0052] 1) Deletion of all or part of the gene encoding the polypeptide;

[0053] 2) modifying the expression control region (or expression control sequence) to reduce the expression of the gene encoding the polypeptide;

[0054] 3) Modifying the amino acid sequence constituting the polypeptide to eliminate or weaken the activity of the polypeptide (for example, deleting / replacing / adding one or more amino acids in the amino acid sequence);

[0055] 4) Modifying a gene sequence encoding a polypeptide to eliminate or weaken the activity of the polypeptide (for example, deleting / replacing / adding one or more nucleotides in the nucleotide sequence of the polypeptide gene to encode the modified polypeptide, thereby eliminating or weakening the activity of the polypeptide);

[0056] 5) modifying the nucleotide sequence encoding the start codon, the Shine-Dalgarno sequence or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0057] 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to a gene transcript encoding a polypeptide;

[0058] 7) adding a sequence complementary to the Shine-Dalgarno sequence of the gene encoding the polypeptide upstream of the Shine-Dalgarno sequence to form a secondary structure that prevents ribosomes from attaching;

[0059] 8) adding a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE); or

[0060] 9) A combination of two or more selected from 1) to 8), but not particularly limited thereto.

[0061] For example,

[0062] 1) Deleting part or all of a gene encoding a polypeptide may be by deleting all of the polynucleotide encoding the endogenous target polypeptide in the chromosome, replacing it with a polynucleotide in which some nucleotides are deleted, or replacing it with a marker gene.

[0063] 2) Modification of the expression control region (or expression control sequence) can be mutation of the expression control region (or expression control sequence) by deletion, insertion, non-conservative substitution or conservative substitution or a combination thereof, or replacement with a sequence having weaker activity. The expression control region includes a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling transcription and translation termination, but is not limited thereto.

[0064] 3) and 4) modification of the amino acid sequence or polynucleotide sequence may be a mutation in the sequence through deletion, insertion, non-conservative substitution or conservative substitution or a combination thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence improved to have weaker activity, or replacement with an amino acid sequence or polynucleotide sequence improved to have no activity, thereby weakening the activity of the polypeptide, but not limited thereto. For example, the expression of a gene may be inhibited or weakened by introducing a mutation in the polynucleotide sequence to form a stop codon, but not limited thereto.

[0065] 5) Modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, replaced with a nucleotide sequence encoding another start codon having a lower polypeptide expression rate instead of the endogenous start codon, but is not limited thereto.

[0066] 6) For the introduction of antisense oligonucleotides (e.g., antisense RNA) that complementarily bind to gene transcripts encoding polypeptides, reference may be made to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1 (1) 1986].

[0067] 7) A sequence complementary to the Shine-Dalgarno sequence of the gene encoding the polypeptide is added upstream of the Shine-Dalgarno sequence to form a secondary structure that prevents ribosomes from attaching, which may make the mRNA unable to be translated or reduce its translation rate.

[0068] In addition, 8) adding a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE) may make the antisense nucleotide complementary to the gene transcript encoding the polypeptide, thereby weakening the activity.

[0069] For example, the activity of the carbamoyl phosphate synthetase of the present invention can be weakened by modifying the start codon of the gene transcript encoding the carbamoyl phosphate synthetase, specifically, it can be weakened by replacing the start codon of the carAB operon gene transcript encoding the carbamoyl phosphate synthetase with another start codon having a lower polypeptide expression rate. For another example, the activity of the carbamoyl phosphate synthetase of the present invention can be weakened by modifying the promoter of the gene encoding the carbamoyl phosphate synthetase, or by replacing it with a weakened promoter, but is not limited thereto.

[0070] The modification of some or all of the polynucleotides in the microorganisms disclosed herein can be induced by: (a) homologous recombination using a vector for chromosome insertion in the microorganism, or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) treatment with light (e.g., ultraviolet light and radiation) and / or chemicals, but not limited thereto. Methods for modifying some or all of the genes may include methods by DNA recombination technology. For example, a nucleotide sequence or vector comprising a nucleotide sequence homologous to the target gene can be introduced into the microorganism to cause homologous recombination, resulting in partial or complete deletion of the gene. The nucleotide sequence or vector to be introduced may include a dominant selection marker, but is not limited thereto.

[0071] The vector of the present disclosure may comprise a DNA construct comprising a nucleotide sequence of a polynucleotide encoding a desired polypeptide, which is operably linked to a suitable expression regulatory region (or expression control sequence) so that the desired polypeptide can be expressed in a suitable host. The expression regulatory region may comprise a promoter capable of initiating transcription, any operon sequence controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling transcription and translation termination. The vector may be transformed into a suitable host cell and then replicated or functioned independently of the host genome, or may be integrated into the genome itself.

[0072] The vector used in the present disclosure is not particularly limited, but any vector known in the art can be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as phage vectors or cosmid vectors; pDZ system, pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0073] For example, the polynucleotide encoding the desired polypeptide can be inserted into the chromosome by the vector for intracellular chromosome insertion. The polynucleotide can be inserted into the chromosome by any method known in the art, such as homologous recombination, but is not limited thereto. The carrier can also include a selection marker for confirming chromosome insertion. The selection marker is used to select cells transformed with the carrier, i.e., for confirming the insertion of the desired nucleic acid molecule, and can use a marker that grants a selectable phenotype (such as drug resistance, auxotrophy, cytotoxic agent resistance or surface polypeptide expression). In the environment of processing with a selection agent, only the cells expressing the selection marker survive or show other phenotypic traits, and therefore the cells transformed can be selected.

[0074] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. As long as the converted polynucleotide can be expressed in the host cell, it can be positioned by being inserted into the chromosome of the host cell or being located outside the chromosome, regardless of the position. In addition, the polynucleotide comprises DNA and / or RNA encoding the desired polypeptide. The polynucleotide can be introduced in any form, as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a gene construct comprising all the elements required for autonomous expression. The expression cassette can generally include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably connected to the polynucleotide. The expression cassette can be in the form of an expression vector capable of self-replication. In addition, the polynucleotide can be introduced into the host cell in its own form and operably connected to the sequence required for expression in the host cell, but is not limited thereto.

[0075] Furthermore, the term "operably linked" means that a polynucleotide sequence is functionally linked to a promoter sequence, which initiates and mediates the transcription of the polynucleotide encoding the desired polypeptide of the present disclosure.

[0076] In another embodiment of the present disclosure, the microorganism of the present disclosure may be a microorganism having L-amino acid production ability.

[0077] The microorganism of the present disclosure may be a microorganism having improved L-amino acid production ability.

[0078] The microorganism of the present disclosure may be a microorganism in which the activity of carbamoyl phosphate synthetase is weakened. In the microorganism, the activity of any one or more of the group consisting of CarA and CarB may be weakened. Specifically, the microorganism of the present disclosure may be a microorganism in which CarA and CarB or carAB operon genes encoding them are weakened; or a microorganism (e.g., a recombinant microorganism) genetically modified to weaken CarA and CarB or carAB operon genes encoding them, but is not limited thereto.

[0079] The microorganism of the present disclosure may be a microorganism naturally having the ability to produce L-amino acids, or a microorganism provided with the ability to produce L-amino acids by weakening carbamoyl phosphate synthetase or a polynucleotide encoding the enzyme in a parent strain that does not have the ability to produce L-amino acids, but is not limited thereto.

[0080] For the purpose of the present invention, the recombinant microorganism of the present invention can be a microorganism that produces L-amino acids by weakening the carbamoyl phosphate synthetase or the polynucleotide encoding the enzyme in the microorganism that produces L-amino acids, compared with the natural wild-type microorganism or the microorganism that produces L-amino acids by comprising carbamoyl phosphate synthetase or the polynucleotide encoding the enzyme, thereby enhancing the L-amino acid production capacity, but is not limited thereto. For example, the unmodified microorganism with unweakened carbamoyl phosphate synthetase (which is the target strain for comparing whether the L-amino acid production capacity is enhanced) can be a wild-type Corynebacterium glutamicum ATCC 13869 strain, a Corynebacterium glutamicum ATCC 13869 strain with weakened ArgF protein activity known as an L-ornithine producing strain, or a Corynebacterium glutamicum ATCC 13869 strain with weakened ArgR protein activity, but is not limited thereto.

[0081] For example, compared to the parent strain before modification or the unmodified microorganism, the recombinant strain with enhanced productivity can have an increase of about 1% or more, specifically, about 2% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 13% or more, about 15% or more, about 16% or more, about 17% or more, about 18% or more, about 19% or more, about 20% or more, about 21% or more, about 24% or more, about 25% or more, about 27% or more, about 30% or more, about 31% or more, about 35% or more, about 40% or more, about 45% or more, about 49% or more. or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 97% or more (the upper limit is not particularly limited, but can be, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 15% or less) of L-amino acid production capacity, but the increase is not limited thereto, as long as the production capacity has a + value increase compared to the production capacity of the parent strain before modification or the unmodified microorganism. In another example, the microorganism with enhanced productivity can have an increase of about 1.01 times or more, about 1.02 times or more, about 1.05 times or more, about 1.06 times or more, about 1.07 times or more, about 1.08 times or more, about 1.09 times or more, about 1.1 times or more, about 1.13 times or more, about 1.15 times or more, about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, about 1.20 times or more, about 1.21 times or more, about 1.24 times or more, about 1.25 times or more, about 1.27 times or more. The L-amino acid production capacity may be about 1.5 times or more, about 1.60 times or more, about 1.65 times or more, about 1.70 times or more, about 1.75 times or more, about 1.80 times or more, about 1.85 times or more, about 1.90 times or more, about 1.95 times or more, or about 1.97 times or more (the upper limit is not particularly limited, but may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less), but is not limited thereto.

[0082] As used herein, the term "unmodified microorganism" does not exclude the bacterial strain comprising a naturally occurring mutation that may occur in the microorganism, and may be a wild-type strain or a natural strain itself, or may be a strain before the genetic variation that is caused due to natural or artificial factors that changes the character. For example, an unmodified microorganism may refer to a bacterial strain wherein the polynucleotides of carbamoyl phosphate synthetase as herein described or encoding this enzyme are not weakened or not yet weakened." unmodified microorganism" may be used interchangeably with "strain before modification", "microorganism before modification", "unchanged strain", "unmodified strain", "unchanged microorganism" or "reference microorganism".

[0083] In the present disclosure, the L-amino acid may be any one or more selected from the group consisting of L-ornithine, L-glutamic acid, L-aspartic acid, and L-phenylalanine.

[0084] In another embodiment of the present disclosure, the microorganism of the present disclosure may be Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium serrata. flavescens), particularly, but not limited to, Corynebacterium glutamicum.

[0085] In another embodiment of the present disclosure, the recombinant microorganism of the present disclosure may be a microorganism having enhanced L-amino acid production capacity by additionally enhancing the activity of some proteins in the L-amino acid biosynthesis pathway or by additionally weakening the activity of some proteins in the L-amino acid degradation pathway.

[0086] Specifically, the microorganism of the present disclosure may be a microorganism in which the activity of ornithine carbamoyltransferase subunit F (ArgF) is additionally attenuated, or the argF gene encoding ArgF is additionally deleted, and / or the activity of arginine repressor protein (ArgR) is additionally attenuated, or the argR gene encoding ArgR is additionally deleted.

[0087] The amino acid sequence of ArgF or ArgR can be obtained from known databases such as Genbank of NCBI. For example, the amino acid sequence of ArgF of the present disclosure may include ANU33618.1 (SEQ ID NO: 19) derived from Corynebacterium glutamicum ATCC 13869 or an amino acid sequence having 90% or higher homology thereto, and the amino acid sequence of ArgR may include ANU33619.1 (SEQ ID NO: 24) derived from Corynebacterium glutamicum ATCC 13869 or an amino acid sequence having 90% or higher homology thereto, but is not limited thereto, and obviously may include proteins having ArgF or ArgR activity derived from various sources.

[0088] However, the reduction of the activity of ArgF and / or ArgR proteins or the deletion of argF and / or argR genes is only an example and is not limited thereto. The microorganism disclosed in the present invention can be a microorganism with enhanced activity of proteins in various known L-amino acid biosynthesis pathways or reduced activity of proteins in degradation pathways.

[0089] As used herein, the term "enhancement" of polypeptide activity refers to the enhancement of the activity of the polypeptide compared to its endogenous activity. Enhancement can be used interchangeably with terms such as activation, upregulation, overexpression, increase, etc. Here, activation, enhancement, upregulation, overexpression and increase can include two situations in which the activity that was not originally present or the activity enhancement is shown compared to the endogenous activity or the activity before modification." Endogenous activity" refers to the activity of the original specific polypeptide of the parent strain or unmodified microorganism before the property change when the property changes due to the genetic variation caused by natural or artificial factors. This can be used interchangeably with "activity before modification". Compared to the endogenous activity, the fact that the activity of the polypeptide is "enhanced", "upregulated", "overexpressed" or "increased" means that the activity of the polypeptide is improved compared to the activity and / or concentration (expression level) of the original specific polypeptide of the parent strain or unmodified microorganism before the property change.

[0090] Enhancement can be achieved by introducing exogenous polypeptides or enhancing the activity and / or concentration (expression level) of endogenous polypeptides. The enhancement of polypeptide activity can be confirmed by an increase in the activity degree and expression level of the corresponding polypeptide or an increase in the amount of the corresponding polypeptide released product.

[0091] In order to enhance the activity of polypeptide, various methods well known in the art can be applied, as long as the activity of the desired polypeptide can be enhanced compared to the microorganism before modification, the method is not limited. Specifically, genetic engineering and / or protein engineering well known to those skilled in the art can be used, which is a conventional method of molecular biology, but the method is not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2, 1-16; Sambrook et al., Molecular Cloning 2012, etc.).

[0092] Specifically, the polypeptides that enhance the present disclosure may be:

[0093] 1) Increasing the copy number of the polynucleotide encoding the polypeptide in the cell;

[0094] 2) Replace the gene expression regulatory region on the chromosome encoding the polypeptide with a more active sequence;

[0095] 3) modifying the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0096] 4) Modify the amino acid sequence of a polypeptide to enhance the activity of the polypeptide;

[0097] 5) modifying a polynucleotide sequence encoding a polypeptide to enhance the activity of the polypeptide (e.g., modifying a polynucleotide sequence of a polypeptide gene to encode a modified polypeptide to enhance the activity of the polypeptide);

[0098] 6) introducing an exogenous polypeptide expressing the polypeptide activity or an exogenous polynucleotide encoding the polypeptide;

[0099] 7) Codon optimization of polynucleotides encoding polypeptides;

[0100] 8) analyzing the tertiary structure of the polypeptide to select and modify or chemically modify exposed sites; or

[0101] 9) A combination of two or more selected from 1) to 8), but not particularly limited thereto.

[0102] More specifically, 1) increasing the number of copies of a polynucleotide encoding a polypeptide in a cell can be achieved by introducing a vector into a host cell, the vector can replicate and function independently of the host, and the polynucleotide encoding the corresponding polypeptide is operably linked to the vector. Alternatively, this increase can be achieved by introducing one copy or two or more copies of the polynucleotide encoding the corresponding polypeptide into the chromosome of the host cell. The introduction into the chromosome can be carried out by introducing a vector capable of inserting the polynucleotide into the chromosome of the host cell, but is not limited thereto. The vector is as described above.

[0103] 2) Replacing the gene expression regulatory region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence showing strong activity may be, for example, a variation in the sequence due to deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacing it with a sequence showing stronger activity, thereby further enhancing the activity of the expression regulatory region. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling transcription and translation termination, etc. For example, the replacement may be replacing the original promoter with a strong promoter, but is not limited thereto.

[0104] Examples of known strong promoters include CJ1 to CJ7 promoters (U.S. 7662943B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. 10584338B2), O2 promoter (U.S. 10273491B2), tkt promoter, yccA promoter, etc., but are not limited to these.

[0105] 3) Modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, replaced with a nucleotide sequence encoding another start codon having a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0106] 4) and 5) modification of the amino acid sequence or polynucleotide sequence may be due to a deletion, insertion, non-conservative substitution or conservative substitution or a combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or substitution with an amino acid sequence or polynucleotide sequence that is improved to exhibit stronger activity, or substitution with an amino acid sequence or polynucleotide sequence that is improved to be more active, thereby enhancing the activity of the polypeptide, but is not limited thereto. The replacement may be specifically performed by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited thereto. The vector used herein may also contain a selection marker for confirming chromosomal insertion.

[0107] 6) The introduction of an exogenous polynucleotide that exhibits polypeptide activity can be to introduce an exogenous polynucleotide into a host cell, and the polypeptide encoded by the exogenous polynucleotide exhibits the same or similar activity as the polypeptide. There is no limitation on its source or sequence, as long as the exogenous polynucleotide exhibits the same or similar activity as the polypeptide. The method used in the introduction can be carried out by appropriately selecting a known transformation method by a person skilled in the art. As the introduced polynucleotide is expressed in the host cell, a polypeptide can be produced, and its activity can be enhanced.

[0108] 7) Codon optimization of a polynucleotide encoding a polypeptide can be codon optimization of an endogenous polynucleotide to enhance transcription or translation in a host cell, or codon optimization of an exogenous polynucleotide to optimize transcription and translation in a host cell.

[0109] 8) Analyzing the tertiary structure of a polypeptide to select and modify or chemically modify an exposed site can be, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing known protein sequence information, determining a template protein candidate according to the degree of sequence similarity, confirming the structure based on this, and modifying or chemically modifying the exposed site to be modified or chemically modified.

[0110] Based on the activity or concentration of the polypeptide expressed in the wild type or microbial strain before modification, this enhancement of polypeptide activity can be an increase in the activity or concentration / expression level of the corresponding polypeptide, or an increase in the amount of the product produced by the corresponding polypeptide, but is not limited thereto.

[0111] Another aspect of the present disclosure provides a method for producing L-amino acids, the method comprising the step of culturing a microorganism of the genus Corynebacterium having an L-amino acid-producing ability in a culture medium, the activity of carbamoyl-phosphate synthetase in the microorganism being attenuated.

[0112] The method for producing L-amino acids disclosed herein may include the step of culturing the following microorganisms in a culture medium: microorganisms in which CarA and CarB or carAB operon genes encoding them are attenuated; or microorganisms genetically modified to attenuate CarA and CarB or carAB operon genes encoding them, as described above.

[0113] As used herein, the term "culture" refers to growing the microorganisms of the present disclosure under appropriately adjusted environmental conditions. The culture procedure of the present disclosure can be carried out according to suitable culture media or culture conditions known in the art. Those skilled in the art can easily adjust this culture procedure according to the selected strain. Specifically, the culture can be batch type, continuous type and / or fed-batch type, but is not limited thereto.

[0114] As used herein, "culture medium" refers to a mixture containing nutrients required for culturing the microorganisms of the present disclosure as the main components, wherein the culture medium provides nutrients including water, growth factors, etc. necessary for survival and growth. Specifically, for the culture medium and other culture conditions for culturing the microorganisms of the present disclosure, any culture medium used for conventional culture of microorganisms can be used without particular limitation. However, the microorganisms of the present disclosure can be cultured in a general culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin, etc. under aerobic conditions, while controlling temperature, pH, etc.

[0115] Specifically, a culture medium of a microorganism of the genus Corynebacterium can be found in the document ["Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington, DC, USA, 1981)].

[0116] In the present disclosure, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steep liquor may be used, and in particular, carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and appropriate amounts of various other carbon sources may be used without restriction. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0117] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc., amino acids such as glutamic acid, methionine, glutamine, etc., and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition product, defatted soybean cake or its degradation product, etc. can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0118] The phosphorus source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and their corresponding sodium salts. The inorganic compound may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc., and may also include amino acids, vitamins and / or suitable precursors. These components or precursors may be added to the culture medium in batches or continuously. However, the present disclosure is not limited thereto.

[0119] In addition, in the process of cultivating the microorganism of the present disclosure, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the culture medium in an appropriate manner. In addition, defoamers such as fatty acid polyethylene glycol esters can be used to suppress foam formation during cultivation. In addition, oxygen or oxygen-containing gas can be injected into the culture medium to maintain the aerobic state of the culture medium, or gas can not be injected, or nitrogen, hydrogen or carbon dioxide gas can be injected to maintain the anaerobic or non-aerobic state of the culture medium, but it is not limited thereto.

[0120] In the culture of the present disclosure, the culture temperature may be maintained at 20° C. to 45° C., specifically, at 25° C. to 40° C., and the culture may be performed for about 10 hours to about 160 hours, but is not limited thereto.

[0121] The L-amino acids produced by the culture of the present disclosure may be released into the medium, or may be retained in the cells.

[0122] The L-amino acid may be any one or more selected from the group consisting of L-ornithine, L-glutamic acid, L-aspartic acid, and L-phenylalanine.

[0123] The method for producing L-amino acids of the present disclosure may further include, for example, before the culturing step, the step of preparing the microorganism of the present disclosure, preparing a culture medium for culturing the microorganism, or a combination thereof (regardless of the order, in any order).

[0124] The method for producing L-amino acids of the present disclosure may further include a step of recovering L-amino acids from a culture medium (a culture medium that has been cultured) or a cultured microorganism. A recovery step may also be included after the culture step.

[0125] Recovery can include: according to the method of culturing microorganisms disclosed herein, such as batch, continuous or fed-batch type culture, collecting the desired L-amino acid by using suitable methods known in the art. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonic treatment, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography, etc.), HPLC and combinations of these methods can be used, and the desired L-amino acid can be recovered from the culture medium or microorganism by using suitable methods known in the art.

[0126] In addition, the method for producing L-amino acids disclosed herein may further include a purification step. Purification may be performed using a suitable method known in the art. In an exemplary embodiment, when the method for producing L-amino acids disclosed herein includes a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously without regard to order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0127] Regarding the methods disclosed herein, carbamoyl-phosphate synthetase, polynucleotides, vectors, microorganisms, etc. are as described in other aspects.

[0128] Another aspect of the present disclosure provides a composition for producing L-amino acid, the composition comprising a Corynebacterium microorganism in which carbamoyl phosphate synthetase is attenuated; a medium for culturing the microorganism; or a combination thereof.

[0129] The composition of the present disclosure may also include any suitable excipients commonly used in compositions for producing L-amino acids, and such excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, etc., but are not limited thereto.

[0130] Another aspect of the present disclosure provides a method for preparing a Corynebacterium microorganism having an L-amino acid-producing ability, the method comprising the step of attenuating carbamoyl-phosphate synthetase.

[0131] Another aspect of the present disclosure provides use of a microorganism of the genus Corynebacterium in which the activity of carbamoyl-phosphate synthetase is attenuated for producing L-amino acids.

[0132] Carbamoyl phosphate synthetase, attenuation, microorganism belonging to the genus Corynebacterium, etc. are as described in other aspects.

[0133] [Mode for carrying out the invention]

[0134] The present disclosure will be described in more detail below by way of exemplary embodiments. However, the following exemplary embodiments are only for illustrating preferred embodiments of the present disclosure, and therefore are not intended to limit the scope of the present disclosure thereto. At the same time, those skilled in the art or similar technical fields to which the present disclosure belongs can fully understand and easily implement technical matters not described in this specification.

[0135] Example 1: Construction of vector

[0136] Example 1-1: Construction of a vector for replacing the carAB promoter

[0137] A vector for replacing the promoter of the carA gene encoding the small subunit of carbamoyl phosphate synthase (CarA) was constructed. The information of the carAB gene (SEQ ID NO:3) encoding CarA (ANU33813.1, SEQ ID NO:1) and its surrounding nucleotide sequence (NZ_CP016335.1) was obtained from NIH GenBank and used to construct a vector for replacing the promoter region of carA with the promoter (SEQ ID NO:4) of betP (ANU33153.1). The genome of wild-type Corynebacterium glutamicum ATCC 13869 was used as a template, and primer pairs SEQ ID NO:5 and 6, primer pairs SEQ ID NO:7 and 8, and primer pairs SEQ ID NO:9 and 10 were used to amplify the betP promoter, homologous recombination A arm, and homologous recombination B arm by PCR (SolTM Pfu-X DNA polymerase), thereby obtaining respective DNA fragments.

[0138] The obtained promoter and recombinant wall gene fragments were cloned into the vector pDCM2 (Korean Patent Publication No. 10-2020-0136813) cut with SalI and BamHI restriction enzymes using Gibson assembly (DG Gibson et al., NATURE METHODS, Vol. 6, No. 5, May 2009, NEBuilder HiFi DNA Assembly Master Mix) to construct the vector. The constructed vector was transformed into Escherichia coli (E. coli) DH5α and plated on LB solid medium containing kanamycin (25 mg / l). In order to select the colonies transformed with the vector, PCR was performed using primers for SEQ ID NOS: 11 and 12, and plasmids were obtained from the selected colonies using a known plasmid extraction method. The obtained plasmid was named pDCM2-PbetP-carA.

[0139] Example 1-2: Construction of a vector for mutating the carA start codon

[0140] A vector for replacing the start codon gtg of the carA gene with ttg was constructed. The genome of wild-type Corynebacterium glutamicum ATCC 13869 was used as a template, and the homologous recombination A arm was amplified using primer pairs SEQ ID NOS: 7 and 13, and the homologous recombination B arm was amplified using primer pairs SEQ ID NOS: 10 and 14. Thereafter, a plasmid was obtained in the same manner as above and named pDCM2-carA (g1t).

[0141] Example 1-3: Construction of a vector for mutating the carB start codon

[0142] A vector was constructed in which the initiation codon atg of the carB gene was replaced with ttg. The information of the carAB gene (SEQ ID NO:3) encoding the large subunit of carbamoyl phosphate synthetase (SEQ ID NO:2) and its surrounding nucleotide sequence was obtained from NIH GenBank (NZ_CP016335.1). The genome of wild-type Corynebacterium glutamicum ATCC 13869 was used as a template, and the homologous recombination A arm and the homologous recombination B arm were amplified using primer pairs SEQ ID NOS:15 and 16 and primer pairs SEQ ID NOS:17 and 18, respectively. Afterwards, a plasmid was obtained in the same manner as above and named pDCM2-carB(a1t).

[0143] The sequences of the primers used in Examples 1-1 to 1-3 are shown in Table 1 below.

[0144] [Table 1]

[0145]

[0146] Example 2: Preparation of strains producing L-ornithine

[0147] Example 2-1: Construction of vector for ArgF inactivation

[0148] In order to prepare a strain producing L-ornithine, a vector was constructed in which the serine at position 55 of the ArgF (ANU33618.1, SEQ ID NO: 19) protein sequence was replaced with a stop codon. The genome of wild-type Corynebacterium glutamicum ATCC1 13869 was used as a template, and the homologous recombination A arm and the homologous recombination B arm were amplified using primer pairs SEQ ID NOS: 20 and 21 and primer pairs SEQ ID NOS: 22 and 23, respectively. Thereafter, a plasmid was obtained in the same manner as above and named pDCM2-argF (S55*).

[0149] Example 2-2: Construction of vector for ArgR inactivation

[0150] A vector was constructed in which the glutamic acid at position 47 of the ArgR (ANU33619.1, SEQ ID NO: 24) protein sequence was replaced with a stop codon. The genome of wild-type Corynebacterium glutamicum ATCC 13869 was used as a template, and the homologous recombination A arm and the homologous recombination B arm were amplified using primer pairs SEQ ID NOS: 25 and 26 and primer pairs SEQ ID NOS: 27 and 28, respectively. Thereafter, a plasmid was obtained in the same manner as above and named pDCM2-argR (E47*).

[0151] The sequences of the primers used in Examples 2-1 to 2-2 are shown in Table 2 below.

[0152] [Table 2]

[0153]

[0154] Example 2-3: Preparation of strains producing L-ornithine

[0155] In order to prepare a strain producing L-ornithine, wild-type Corynebacterium glutamicum ATCC 13869 was transformed by electroporation (Appl. Microbiol. Biotechnol. (1999) 52: 541-545) with the pDCM2-argF (S55 *) vector constructed in Example 2-1, and then a second exchange process was performed to obtain a strain in which the nucleotide sequence at position 164 of argF was replaced by adenine (a) by cytosine (c), so that position 55 of the protein sequence was replaced by a stop codon. PCR and sequencing analysis were performed using primers SEQ ID NOS: 20 and 23, which were able to amplify the adjacent region containing the corresponding gene insertion position and confirmed the corresponding genetic manipulation. The strain thus obtained was named C.gl::argF *.

[0156] In order to prepare a strain with further improved ornithine production in C.gl::argF*, the pDCM2-argR(E47*) vector prepared in Example 2-2 was used to obtain a strain in the same manner as above. PCR and sequencing analysis were performed using primers SEQ ID NOS: 25 and 28, which were able to amplify the adjacent region containing the corresponding gene insertion position, and confirmed the corresponding genetic manipulation. The strain thus obtained was named C.gl::argF*_argR*.

[0157] Example 3: Preparation of CarAB-weakened strains

[0158] The PDC 2-pbe TP-carA and PDC 2-carA (g1t) vectors constructed in Examples 1-1 to 1-2 were transformed into wild-type Corynebacterium glutamicum ATCC 13869 and C.gl::argF* and C.gl::argF*_argR* prepared in Example 2-3 by electroporation, and strains were obtained by the second exchange process. SEQ ID NOS: 6 and 9 were subjected to PCR and sequencing analysis using primers, and the corresponding genetic manipulation was confirmed. The strains thus obtained were named in the following order: C.gl::PbetP-carA, C.gl::carA (g1t), C.gl::argF*_PbetP-carA, C.gl::argF*_carA (g1t), C.gl::argF*_argR*_PbetP-carA, C.gl::argF*_argR*_carA (g1t).

[0159] In order to prepare a strain in which the carB promoter and the start codon are replaced, the plasmid pDCM2-carB (a1t) vector obtained in Example 1-3 is transformed into wild-type Corynebacterium glutamicum ATCC 13869, C.gl::argF*, C.gl::argF*_argR* by electroporation, and the strain is obtained by the second exchange process. PCR and sequencing analysis were performed using primers SEQ ID NOS: 16 and 19, which can amplify the adjacent region containing the corresponding gene insertion position and confirm the corresponding genetic operation. The strains thus obtained are named in the following order: C.gl::carB (a1t), C.gl::argF*_carB (a1t), C.gl::argF*_argR*_carB (a1t).

[0160] Example 4: Evaluation of L-amino acid production capacity of recombinant strains

[0161] The C.gl::PbetP-carA, C.gl::carA(g1t), C.gl::carB(a1t) strains prepared in Example 3 and a control wild-type Corynebacterium glutamicum strain were cultured to compare cell quality and L-amino acid production capacity.

[0162] First, each strain was inoculated into a 250 ml corner-baffled flask containing 25 ml of seed medium and cultured at 30° C. with shaking at 200 rpm for 16 hours. 1 ml of seed culture was inoculated into a 250 ml corner-baffled flask containing 24 ml of production medium and cultured at 33° C. with shaking at 200 rpm for 40 hours.

[0163] After the cultivation was completed, the production of 20 kinds of L-amino acids was measured using HPLC.

[0164] <Seed culture medium (pH 7.0)>

[0165] 20g glucose, 10g peptone, 5g yeast extract, 1.5g urea, 4g KH 2 PO 4 8g K 2 HPO 4 、0.5gMgSO 4 7H 2 O, 0.1 mg biotin, 1 mg thiamine HCl, 22 mg calcium pantothenate, 2 mg niacinamide (based on 1 liter of distilled water)

[0166] <Production medium (pH 7.0)>

[0167] 50g raw sugar, 25g (NH 4) 2 SO 4 , 1g yeast extract, 0.55g KH 2 PO 4 、0.6 g MgSO 4 7H 2 O, 0.9 mg biotin, 4.5 mg thiamine HCl, 4.5 mg calcium pantothenate, 30 mg niacinamide, 9 mg MnSO 4 , 9mg FeSO 4 , 0.45mg ZnSO 4 , 0.45 mg CuSO 4 、30g CaCO 3 (Based on 1 liter of distilled water)

[0168] The above experiment was repeated three times, and the culture results (average values) are shown in Table 3. The increase rate of each L-amino acid concentration was calculated as (experimental group-control group) / control group.

[0169] [Table 3]

[0170]

[0171] As shown in Table 3, the C.gl::PbetP-carA, C.gl::carA(g1t), and C.gl::carB(a1t) strains showed increased production and yield of L-glutamic acid, L-aspartic acid, and L-phenylalanine compared to the control wild-type Corynebacterium glutamicum ATCC 13869 strain.

[0172] Example 5: Evaluation of L-ornithine production capacity of recombinant strains

[0173] The C.gl::argF*_PbetP-carA, C.gl::argF*_carA(g1t), C.gl::argF*_carB(a1t), C.gl::argF*_argR*_PbetP-carA, C.gl::argF*_argR*_carA(g1t), C.gl::argF*_argR*_carB(a1t) strains prepared in Example 3, as well as the control strains C.gl::argF* and C.gl::argF*_argR*, were cultured in the same manner as in Example 4 using the following culture medium to compare cell quality and L-ornithine production capacity.

[0174] <Seed culture medium (pH 7.0)>

[0175] 20g glucose, 10g peptone, 5g yeast extract, 1.5g urea, 4g KH 2 PO4 8g K 2 HPO 4 、0.5gMgSO 4 7H 2 O, 0.1 mg biotin, 1 mg thiamine HCl, 22 mg calcium pantothenate, 2 mg niacinamide (based on 1 liter of distilled water)

[0176] <Production medium (pH 7.0)>

[0177] 50g raw sugar, 25g (NH 4 ) 2 SO 4 , 1g yeast extract, 0.55g KH 2 PO 4 、0.6 g MgSO 4 7H 2 O, 0.2g L-arginine, 0.9mg biotin, 4.5mg thiamine HCl, 4.5mg calcium pantothenate, 30mg niacinamide, 9mg MnSO 4 , 9mg FeSO 4 , 0.45mg ZnSO 4 , 0.45 mg CuSO 4 、30g CaCO 3 (Based on 1 liter of distilled water)

[0178] The above experiment was repeated three times, and the culture results (average values) are shown in Table 4. The L-ornithine yield was calculated as ornithine concentration / sugar consumption, and the concentration increase rate was calculated as (L-ornithine concentration produced by the experimental group-L-ornithine concentration produced by the control group (wild-type Corynebacterium glutamicum ATCC 13869 strain)) / L-ornithine concentration produced by the control group (wild-type Corynebacterium glutamicum ATCC 13869 strain).

[0179] [Table 4]

[0180]

[0181] As shown in Table 4, compared with the control strain C.gl::argF*, C.gl::argF*_PbetP-carA, C.gl::argF*_carA(g1t) and C.gl::argF*_carB(a1t) strains showed increased production capacity and yield. Similarly, compared with the control strain C.gl::argF*_argR*, C.gl::argF*_argR*_PbetP-carA, C.gl::argF*_argR*_carA(g1t), C.gl::argF*_argR*_carB(a1t) strains showed increased production capacity and yield.

[0182] Based on the above description, it will be appreciated by those skilled in the art that the present disclosure may be implemented in other specific forms without departing from its technical spirit or essential features. In this regard, the above embodiments should be understood to be illustrative rather than restrictive in all respects. The scope of the present disclosure should be understood as the meaning and scope of the appended claims rather than detailed description, and all changes or modifications derived from equivalent substitutions fall within the scope of the present disclosure.

Claims

1. A Corynebacterium microorganism having an L-amino acid-producing ability, wherein the activity of carbamoyl-phosphate synthetase is attenuated.

2. The microorganism according to claim 1, wherein the L-amino acid is any one or more selected from the group consisting of L-ornithine, L-glutamic acid, L-aspartic acid and L-phenylalanine.

3. The microorganism according to claim 1, wherein the activity of any one or more selected from the group consisting of a carbamoyl-phosphate synthetase small subunit (CarA) and a carbamoyl-phosphate synthetase large subunit (CarB) is attenuated. The microorganism according to claim 1 , wherein the carbamoyl-phosphate synthetase is encoded by a carAB operon gene. 5 . The microorganism according to claim 3 , wherein CarA and CarB have the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively, or amino acid sequences having 90% or more homology thereto. The microorganism according to claim 1 , wherein the activity of ornithine carbamoyltransferase subunit F (ArgF) is further attenuated. The microorganism according to claim 1 , wherein the activity of arginine repressor (ArgR) is further attenuated. The microorganism according to claim 1 , wherein the Corynebacterium microorganism is Corynebacterium glutamicum .

9. A method for producing L-amino acids, the method comprising the step of culturing a microorganism of the genus Corynebacterium having an L-amino acid-producing ability in a culture medium, wherein the activity of carbamoyl-phosphate synthetase in the microorganism is attenuated.

10. The method according to claim 9, wherein the L-amino acid is any one or more selected from the group consisting of L-ornithine, L-glutamic acid, L-aspartic acid and L-phenylalanine.

11. The method according to claim 9, wherein the activity of ornithine carbamoyltransferase subunit F (ArgF) is further attenuated in the microorganism.

12. The method according to claim 9, wherein the activity of arginine repressor (ArgR) is further attenuated in the microorganism.

13. A composition for producing L-amino acids, the composition comprising a microorganism of the genus Corynebacterium in which carbamoyl-phosphate synthetase activity is attenuated; a culture medium for culturing the microorganism; or a combination thereof.

14. Use of a microorganism of the genus Corynebacterium in which carbamoyl-phosphate synthetase is attenuated for producing L-amino acids.

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